Cryoimmobilization followed by freeze substitution gives vastly improved ultrastructure and preservation of antigenicity in most cell types when compared to traditional processing methods. Presently there is no high-pressure freezer (HPF) unit available to users of the Biological Imaging Facility (BIF) at Northwestern University. Funds are requested to purchase a Leica EM-PACT2 High-Pressure Freezer (HPF) and Automated Freeze-Substitution (AFS2) system, the most up-to-date cryopreparation equipment. We will use the HPF to: 1) achieve ultrastructural preservation as close to the sample's native state as possible, 2) immunolocalize gene products with reduced mobility at high resolution, and 3) characterize mutant phenotypes at the EM level. The EM-PACT2 system can freeze samples up to about 600mm thick with a 200mm depth of high quality freezing. This cryoprocessing system provides reliable freezing using specimen holders that can accommodate a wide variety of specimen types. With this system we can investigate many biological systems (and many materials specimens) that were previously inaccessible. We will use this system in a variety of developmental and cell biological studies including: 1) estrogen regulation of presynaptic vesicles at brain synapses, 2) investigation of the pathways leading to inhibin A and B secretion from granulosa cells, 3) the organization of viral proteins and necessary host cell factors at the site of viral budding, 4) the hydrodynamics of aqueous humor outflow, 5) the role of BRCA2 and centrobin in the regulation of centrosome duplication in cultured tumor cells, and 6) ADDL-induced synaptic spine changes in primary hippocampal cultures. These projects will significantly benefit from the ability of HPF-AFS to stop cellular activity and restrict cell component relocation almost instantaneously, features that conventional EM preparation methods do not offer. These projects require a cryoprocessing system with the greatest flexibility, ease of use, and consistency of results. Based on the experience of the manager and on demonstrations of the two available cryoprocessing units, we have determined that the Leica EM-PACT2/AFS2 system best suits our needs. This equipment will be housed in BIF on the Northwestern University Evanston Campus. BIF is a shared-use facility available to Northwestern researchers, neighboring academic and health care institutions, and outside individuals. All of the major users listed in this proposal are either currently funded by NIH or have proposals pending. Relevance: Results obtained from these studies will have significant impact on public health research in the areas of epilepsy, infertility, cancer, viral transmission, glaucoma, and Alzheimer's disease. [unreadable] [unreadable] [unreadable]